The invention relates to ganoderma spore powder refined polysaccharide with significant auxiliary anti-tumor activity and a preparation method and application thereof, and belongs to the field of medicinal-fungus extraction and separation and the field of drug and food application development. The refined polysaccharide is mainly prepared from heteropolysaccharide composed of mannose, glucose, galactose and fucose according to the molar ratio of 1: (40-50): (4-5.5): (1.5-2), the purity of the refined polysaccharide is greater than 80%, and the weight-average molecular weight is 160-200 KD. Thepreparation method of the ganoderma spore powder refined polysaccharide includes the steps of using hot water for dynamic extraction, removing residues by combining sedimentation and rough filtration, conducting refined filtration and clarification, removing impurities through ultrafiltration-membrane concentration, and drying through spray or airing through alcohol precipitation. The preparationmethod of the ganoderma spore powder refined polysaccharide is environmentally friendly and smart, saves energy, can solve industrialized difficulties and has significant scale advantages, prepared products meet the requirements, about the safety, availability, controllability and stability, in a drug and medicine quality controlling system. The ganoderma spore powder refined polysaccharide has significant functional activity at the auxiliary anti-tumor aspect and can be used for developing tumor prevention and treating drugs, and development of food and healthcare products in related fieldscan be carried out.